The theme of project 1 is the characterization of a novel class of adjuvants and is based on our discovery that mammalian cells contain molecules with unique adjuvant properties that are released when cells are injured and alert the immune system to danger. Our underlying hypothesis is that these endogenous molecules normally play an important role in initiating immune responses to infections and can be exploited as a novel class of adjuvants for vaccines. The objectives of this project are to establish the molecular identify of these molecules, to elucidate their biological effects and underlying mechanisms of action, and to test their potential utility for vaccines for the class A biodefense pathogen, LCMV. An important aspect of these studies will be our interaction with projects #2 and #3 to examine whether the endogeous adjuvant works through Toll-like receptors (TLR) and/or whether various TLR agonists synergize with stimulation by the endogenous adjuvants. This project consists of three Aims. The first Aim will purify the major endogenous adjuvants and determine their molecular structure. The second Aim will elucidate the kinds of immune responses they enhance, determine how they compare to TLR agonists and evaluate whether these molecules are useful alone or in combination as vaccine adjuvants. The importance of this goal is that it will define what kinds of immune responses these adjuvants help to regulate and hence the settings in which they will be useful for enhancing vaccine efficacy. The third aim will elucidate the cellular and molecular mechanisms of the endogenous adjuvant activity. We will examine the hypothesis that they augment immune responses by acting on dendritic cells and define how dendritic cell function (or that of other cellular targets) is changed. We will also test the hypothesis that these adjuvant molecules signal through TLRs. In addition, we will compare the molecular changes induced by the endogenous adjuvants to those stimulated by microbial agonists of TLRs. Together these Aims and the ones of the other projects will provide insight into how infection with LCMV leads to immunity versus disease and identify novel classes of adjuvants for vaccine development.